PROJECT SUMMARY/ABSTRACT Uterine fibroids (UFs; leiomyomas) are the most important benign neoplastic threat to women's health worldwide, but disproportionately affect women of color, particularly African American (AA) women, who have a threefold higher incidence rate and relative risk of UFs than Caucasian (CC) women. While the underlying cause for this risk disparity is not fully understood, recent studies implicate hypovitaminosis D as a major contributor. Thus, AA women have a tenfold increased risk of vitamin D deficiency compared to CC women, and as we first reported, UF risk is inversely correlated with 25-hydroxy vitamin D serum levels. Nonetheless, it is not clear whether and how the processes that drive UF formation and determine relative risk are genetically or biochemically linked. In this regard, we and others have identified somatic mutations in the transcriptional Mediator subunit MED12 as the dominant drivers of UFs, accounting for ~70% of tumors. Notably, MED12- mutant UFs are characterized by significant chromosomal loss and rearrangement, suggesting genomic instability as a driving force in tumor progression. Herein, we clarify the molecular basis for mutant MED12- driven genomic instability, and further identify vitamin D3 receptor signaling as a likely suppressor of this process. We show that MED12-mutant UF stem cells (SCs) accumulate high levels of unrepaired DNA double- strand breaks (DSBs) through downregulation of key DNA damage response (DDR) and repair genes. Notably, we find the vitamin D3/receptor axis to be a variable modulator of MED12-regulated DDR gene expression. Thus, we show that reduced vitamin D3/receptor signaling suppresses, while elevated signaling activates, DDR genes downregulated in MED12-mutant UF SCs. Based on these findings, we hypothesize that hypovitaminosis D exacerbates DNA damage accumulation and genomic instability arising in MED12-mutant UFs, leading to enhanced tumor progression and burden. Accordingly, we propose that vitamin D3, through reparation of an impaired DDR will provide therapeutic benefit in MED12-mutant tumors. To test these hypotheses, we will (1) Elucidate the molecular basis of genomic instability in MED12-mutant UFs. We will determine if DSB accumulation in MED12-mutant UF SCs derives from defects in DNA damage-induced checkpoint signaling and repair and/or R-loop-induced replication stress. (2) Investigate the relationship between vitamin D3 and MED12 in UF genome maintenance. We will ask whether and how vitamin D3 signaling strength modulates the DDR defects in MED12-mutant UF SCs, relate this activity to patient race and serum vitamin D levels, and elucidate the mechanism by which the vitamin D3/receptor axis and MED12 coordinately control the DDR network at the genomic and epigenomic levels; (3) Examine the therapeutic potential of vitamin D3 in a preclinical mouse model of human UFs. Using a renal capsule mouse model of human UFs, we will evaluate vitamin D3 and its potent non-hypercalcemic analogs for therapeutic efficacy, safety, and mechanism of action, including impact on tumor DNA damage load and DDR gene networks.